1. Field of the Invention
The present invention relates to improvements in methods and apparatus for preparing cell samples, e.g., human blood samples, for intracellular assay using flow cytometric techniques.
2. Discussion of the Prior Art
Cell surface immunophenotyping using fluorescent flow cytometry has become a relatively routine process for differentiating and counting cells of interest in a cell sample containing many different cell types. Typically, cell surface probes, e.g., fluorochrome-labeled monoclonal antibodies (MABs) or other suitably labeled ligands, specific to antigens on the outer surface of the cells of interest, are used to selectively tag or “stain” such cells for subsequent detection. The flow cytometer operates to detect the stained cells by irradiating individual cells in the sample, one-by one, with radiation specially adapted to excite the fluorochrome labels. When irradiated, the labels fluoresce and their associated cells scatter the incident radiation in a pattern determined by the physical and optical characteristics of the irradiated cell. Suitable photo-detectors within the flow cytometer detect the scattered radiation and fluorescence, and their respective output signals are used to differentiate the different cell types on the basis of their respective light-scattering and fluorescence signatures.
The process for preparing samples for cell surface immunophenotyping is relatively simple. Basically, the cell surface probes are mixed with the cell sample, and the resulting mixture is incubated for a time sufficient to enable the probes to bind to the cells of interest. Thereafter, if the cell sample is a whole blood sample, the tagged cell sample may be lysed to eliminate red blood cells. Optionally, the tagged cell sample may be washed to eliminate interferants, e.g., unattached probes, cell fragments and other debris that may interfere with the detection of the cells of interest. While such sample preparation is often performed manually, automated sample preparation instruments are commercially available for facilitating the sample-preparation process. These instruments are advantageous in that they remove many potential human errors and other variations from the sample-preparation steps they perform, thereby providing more repeatable results. Such automated instruments include, for example, various pipetting instruments that operate under the control of a suitably programmed microprocessor to automatically pipet and dispense precise volumes of sample and reagent materials (e.g., cell-surface markers) into one or more reaction vessels or tubes where the materials are mixed together (e.g., by vibration or vortex mixing). One such instrument is the PrepPlus2™ Sample Prep Instrument made and sold by Beckman Coulter, Inc., Miami, Fla. Other stand-alone instruments that facilitate the sample-preparation process for cell surface immunophenotyping are those that operate to lyse a whole blood sample, e.g. the Q-Prep™ Sample Prep Work Station line of instruments sold by Beckman Coulter, and those that, in effect, wash the sample to eliminate interferants and cellular debris that adversely effect the detection of the cells of interest within the sample. Such sample washing instruments include various centrifuging instruments, as well as the CellPrep™ Cell Washer, also sold by Beckman Coulter, Inc. The latter instrument operates to filter a cell sample presented to it using a microporous (i.e., semi-permeable) hollow fiber membrane, as described in detail in the commonly assigned U.S. Pat. Disclosure No. 2002/0123154 A1 in the names of Burshteyn et al., published on Sep. 5, 2002.
In addition to the many different stand-alone, sample-preparing instruments that are adapted to automatically perform different portions of a cell sample preparation process for cell surface immunophenotyping, there are some flow cytometers that operate to automatically carry out all of the requisite sample preparation steps within the environs of the flow cytometer itself. See, for example, the Cell Dyne™ 4000 Blood Analyzer made by Abbott Laboratories, and the R-1000 Reticulocyte Analyzer made by Toa Electronics. Both of these instruments are capable of providing hematology and fluorescent flow cytometry results on a cell sample prepared within the measuring instrument. Note, in the latter instrument, cell samples containing reticulocytes are prepared for flow cytometery analysis by mixing the cells sample with a fluorescent stain that is selectively absorbed through the reticulocyte membrane and taken up by the RNA contained by such membrane. In neither of these instruments is the cell sample washed prior to passing the sample through an optical flow cell for analysis.
While cell sample preparation may be regarded as a relatively simple matter in the case of cell surface immunophenotyping and, thus, is readily susceptible to a certain degree of automation, the process for preparing cell samples for intracellular assays is considerably more complex. Such assays, of course, require access to the cell interior so that the intracellular antigens or molecules of interest, e.g., cytokines, chemokines, defensins, effector molecules, etc., can be tagged for identification. Since the probes normally used to tag intracellular antigens and molecules for identification on a flow cytometer are relatively large in size, usually being in the form of a ligand/fluorochrome conjugate, the sample preparation process must include a permeabilization step by which correspondingly large openings are created in the cell membrane to enable passage of these probes into the cell interior. (Note, while reticulocyte assays of the type noted above may be viewed as an intracellular assay in that the reticulocyte's internal RNA, an intracellular antigen, is tagged to identify the reticulocyte, preparation of cell samples for this assay do not require any special pre-processing (e.g. permeabilizing) in order for the relatively small fluorescent dye molecules used to tag the RNA to pass through the pores of the cell membrane and thereby gain access to the RNA within the cell.)
In addition to the need to permeabilize the cell membrane, the sample-preparation process for intracellular assays also requires that cell membranes and contents of the cell be “fixed” prior to the permeabilization step in order to maintain the integrity of the membrane during permeabilization and to prevent the intracellular material from diffusing out of the cells after permeabilization. This fixing step also serves to preserve the localization of the intracellular antigens of interest so that they are more readily accessible to the probes that are about to enter the cells. It will be appreciated that the fixation step cannot be so severe as to (a) prevent the probes from entering the cell membrane, (b) prevent the probes from gaining access to the intracellular antigens of interest, or (c) alter the confirmation of the antigen to the extent that it is unrecognizable by the probe.
A further complicating factor to the intracellular sample-preparation process is the need to ultimately produce a sample that is virtually free of interferants or contaminants that may act to raise the background level against which the antigens of interest is to be detected. It will be appreciated that many intracellular antigens are present in very low amounts within a cell and thus require correspondingly low, substantially interferant-free, background levels to detect. Further, the intracellular antigens of interest are sometimes found in a very low percentage of the cells comprising the analysis sample and thus may be regarded as “rare events.” Hence, controlling the levels of all contaminants in the sample is critical to the accuracy and repeatability of such assays. Due to the need to both fix and permeabilize the cells of interest in preparation for an intracellular assay, the sample-preparation process, as practiced heretofore, has always required multiple wash steps for eliminating the aforementioned interferants that can compromise the detection of the intracellular antigens of interest.
To date, each of the above-noted cell-washing steps associated with the conventional intracellular sample-preparation process has been carried out with a centrifuge. Upon adding a buffer solution to the sample vessel to increase its volume, the sample is spun down at a relatively high g force (e.g., 300-500 g) to produce a concentrated pellet of the cells of interest. A portion of the remaining supernatant containing the interferants and the like is then carefully poured off, and the remaining supernatant is used to re-suspend the sample cells. Rigorous vortex mixing is then necessary to disperse the individual cells of the pellet. While this method of washing cells may be considered as highly effective in physically separating the cells of interest from the soluble components within the reaction vessel, there are some obvious disadvantages associated with centrifugation. For example, it is well known that the removal of any supernatant resulting from a centrifugation process can result in a significant loss in the total number of cells in the sample, or may result in a selective loss of certain cell types. Also, the centrifugation apparatus itself is often relatively bulky and requires substantial laboratory space to accommodate it. Further, in the case of separating permeabilized cells from interferants by centrifugation, there are additional problems. For example, it is known that permeabilized cells become more buoyant as a result of the permeabilizing process; further, the cells also become sticky and tend to clump together. This increase in buoyancy requires centrifugation at greater g forces and for longer periods of time to achieve the desired pelleting. The increased g forces, coupled with the stickier permeabilized membranes, make re-suspension of the individual cells even more difficult. Worse yet, the increased g force can disrupt the cell membrane and the entire cell will be lost.
As may be appreciated from the discussion above, the conventional process for preparing cell samples for intracellular assaying is relatively complex and, owing to its complexity, it is routinely performed manually in a variety of ways. Referring to FIG. 1, the typical conventional process may be summarized as follows: First, a predetermined volume of cell surface markers (e.g., suitably labeled monoclonal antibodies) is added to a predetermined volume of a cell sample (e.g., a whole blood sample) in a reaction vessel. The surface markers are intended to identify, by means of flow cytometry, those cells in which intracellular antigens or molecules are to be assayed. After gently mixing the blood sample and cell surface makers and incubating the mixture for a predetermined time and at a desired temperature (e.g., room temperature), a predetermined volume of a cell-fixing reagent (e.g., a formaldehyde solution) is added. Upon mixing the fixative with the blood sample, which can be achieved by either introducing the fixative into the vessel at a predetermined rate, or by vortexing the fixative and sample after adding the fixative at a much slower rate, the resulting mixture is incubated again. As indicated above, the incubation time and fixative strength are critical to achieve the desired degree of fixation. Thereafter, the fixed sample is washed via centrifugation to rid the sample of interferants. To prepare the sample for centrifugation (washing), an excess of (e.g., 20-fold) buffer solution (e.g., a phosphate-buffered saline solution) is added. The buffer has the effect of quenching the fixing action of the fixative, diluting interfering reagents, and increasing the sample volume to a level suitable for centrifugation. The sample is then transferred to a centrifuge where it is spun down to produce a relatively concentrated pellet of cells covered by a liquid supernatant containing interferants. Upon removing the reaction vessel from the centrifuge and carefully removing (to avoid the loss of cells of interest) most of the supernatant, the remaining materials are subjected to a rigorous vortex mixing, preferably carried out on a suitable mixing instrument, to disperse and re-suspend the cells of the pellet. Then, the intracellular probes (e.g. MAB's with fluorochrome labels) can be added, followed by a predetermined volume of a permeabilizing/lysing reagent (e.g., saponin), or vice versa. After a gentle mixing step, the probes and permeabilizing/lysing reagent are incubated with the fixed sample cells for a predetermined time, and another relatively large volume of buffer is added to quench the permeabilizing and lysing action, to dilute the interfering reagents and to increase the sample volume to a level suitable for another centrifugation. Upon centrifuging the sample again, removing the supernatant and re-suspending the pelleted cells by adding another volume of buffer and vortex mixing the sample, the cell sample is now ready to be transported to the flow cytometer for analysis.
From the above, it will be appreciated that the conventional process for preparing cell samples for intracellular assaying on a flow cytometry is subject to considerable error and non-repeatability. No matter how precise the protocol for carrying out this relatively complex and lengthy process, the need for human involvement can lead to huge variations in the sample produced. Any variations in timing or techniques in aspirating, mixing and dispensing the samples and reagents, either from day-to-day by the same technician, or from technician-to-technician, or from laboratory-to-laboratory, can result in meaningless data, especially in light of the relative rarity or dimness of the events being assayed. Further, the potential for cell loss and cell damage during each of the multiple centrifugation (wash) steps and vortex mixing steps are obvious disadvantages to the present sample preparation process. Clearly, there is a need for a simpler process, one with substantially fewer steps, and one that lends itself to total automation.